4Department of Physiology and Cell Biology, University of Health Sciences, Lahore, Pakistan

*Corresponding Address:
Reproduction Group
Department of Microbiology and Parasitology
Medical School
University of Antioquia
Antioquia
Colombia
Email:wdario.cardona@udea.edu.co

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Abstract

Background

The quality of semen depends upon several factors such as environment,
life style, physical activity, age, and occupation. The aim of this study was to analyze and
compare the conventional and functional semen parameters in men practicing vigorous
physical activity to those of sedentary men.

Materials and Methods

In this descriptive cross-sectional study, semen samples of 17
physically active men and 15 sedentary men were collected for analysis. Semen analysis
was performed according to the World Health Organization (WHO) guidelines, while
functional parameters were evaluated by flow cytometry.

Results

Results showed that several semen parameters (semen volume, viability, progressive motility, total motility, normal morphology, and moribund cells) were superior
in the physically active group in comparison with the sedentary group. Semen parameters
such as viability, progressive motility and total motility, as well as the percentage of moribund spermatozoa were significantly different between both groups. However, sperm
DNA damage, lipid peroxidation and mitochondrial potential were not significantly different among the groups.

Conclusion

Nevertheless, the physical activity shows better semen parameters than sedentary group. Taken together, our results demonstrate that regular physical activity has
beneficial impact in sperm fertility parameters and such a life style can enhance the fertility status of men.

Introduction

The conventional semen analysis involves the
macroscopic (volume, pH, and colour) and microscopic
(motility, concentration, viability, and morphology)
examination (1). It reflects the secretory
activity of the testes, epididymis and accessory sex
glands indirectly (2). Although conventional semen
analysis provides both quantitative and qualitative information, it does not include evaluation of the
functional properties of spermatozoa (3-7). Furthermore,
oxidative stress which may directly contribute
to the origin of male infertility, is not measured
(8). Oxidative stress occurs due to the imbalance
between the reactive oxygen species (ROS), reactive
nitrogen species (RNS), and seminal antioxidant
reserve in the male reproductive tract (9, 10).
These ROS or RNS are produced during normal
cellular metabolism and can be from either endogenous
(normally produced by oxidative phosphorylation
in mitochondria) or exogenous origin (e.g.
produced by leukocytes) (10, 11).

Physiological levels of ROS exert a critical role
in spermatozoa, triggering and mediating important
signaling events to acquire essential functions such
as hyperactivation, capacitation, and acrosome reaction
(10-12). However, an excess in ROS levels is
detrimental to cellular function and spermatozoa are
highly susceptible to oxidative stress due to a lack in
repair mechanisms (8, 13). This may result in damage
to the structural components of the axoneme
which may impact on the motility patterns (14, 15).
It may also induce lipid peroxidation of cellular
membranes (16), thereby disrupting the fluidity of
mitochondrial and plasma membranes (12, 17) and
furthermore lead to oxidative damage to proteins
involved in the fusing of the spermatozoon with the
oolemma (15). Additionally, ROS may cause DNA
damage due to impaired histone remodeling during
sperm maturation (12). Oxidative damage to spermatozoa
has been related with recurrent pregnancy
loss (13, 18, 19) and male infertility (5, 20).

It is well known that certain environmental factors
including prolonged and continued exposure
of the whole body, testes or scrotum to: i. Increased
temperature, even at 37°C (21-23), ii. Environmental
pollutants and endocrine disruptors (21,
24), iii. Electromagnetic radiation (21, 25), as well
as lifestyle factors such as smoking, recreational
drug use, alcohol consumption, obesity and sedentary
occupation or lifestyle (25-29), may influence
sperm quality and male fertility potential (21,
23, 25) mediated by induction of oxidative stress
leading to cell apoptosis. Among several life style
factors, sedentarism have been found associated
with several medical conditions and considered
as one of the main causes of major public health
issues at present (30). According to the definition
of Bernstein et al. (31), individuals are considered
sedentary when they spend less than 10% of their
daily energy expenditure on performing moderate
to vigorous-intensity activities. Also a sedentary
person frequently spends much time sitting or lying
down and performing activities usually associated
with this low energy consumption state such
as sleeping or watching television. Also it is commonly
avoiding any form of exercise or sporting
activities (32). Over the past five decades, changes
in the occupational activities and leisure time have
promoted the sedentary behavior and impacted on
lifestyle (33, 34). As is true for other medical conditions
(obesity and heart diseases), this phenomenon
is equally deleterious for semen quality (35).

On the other hand; physical activity has beneficial
effect on human health and is defined as any voluntary
and repetitive body movements produced by skeletal
muscle action that substantially increases energy
expenditure above the basal state (34, 36). It may be
included in the occupational activities or have diverse
purposes like being aerobic training or training
strength, flexibility and balance, therefore it encompasses
exercise and sport (37, 38). Physical activity
is classified according to the intensity with which it
is practiced and may be quantified in terms of the energy
expenditure as a multiple of the resting metabolic
rate (39). Using the Metabolic Equivalent of the Task
(MET, a physiological measure expressing the energy
cost of physical activities), the moderate physical
activity producing noticeable accelerated heart rate,
ranges from 3.0 to 6.0 MET while the vigorous physical
activity, demanding greater physical effort causing
rapid breathing and a substantial increase in heart rate,
are all the physical activities above 6.0 MET (38, 39).
Some studies have reported a positive relationship (27,
33, 40-44), while others reported a negative one (45-
47) between the practice of physical activity and the
semen quality. Others report no impact of physical activity
on sperm quality (48-51), therefore, the effect of
a physically active lifestyle to improve semen quality
is still controversial (52). The present study was conducted
with the specific aim to evaluate and compare
the semen parameters, conventional as well as functional,
of men practicing vigorous physical activity to
those having a sedentary life style.

Materials and Methods

In this descriptive cross-sectional study, thirty
two men of reproductive age (physically active
group 27.5 ± 6.0 and sedentary group 26.6 ± 5.3
Lalinde-Acevedo et al. years) from Medellin, Colombia were included. The
inclusion criteria were: healthy men, without testicular
disease, with a body mass index (BMI)<26
kg/m2, and those who followed the same lifestyle
pattern for the 12 months preceding the study, it
is, either be physically active group (PAG, practice
vigorous physical activities with a >6 MET
for more than 2 hours per occasion at least 3 times
per week; activities included are cycling, stationary
cycling, calisthenetics, weightlifting, dancing,
running, marital arts, football and swimming) or be
sedentary group (SG, minimal physical activity ≤3
MET, do not practice sportive activities) (39, 53)
(Table 1,). Recreational drug and anabolic steroid
users, smokers or medicated men were excluded
from the study. Ethical approval was obtained from
the Research Ethics Committee of the University of
Antioquia and all patients gave informed consent.
Semen samples were collected from volunteers between
January and July 2014 by masturbation after
a recommended ejaculation abstinence of 3-6 days.
In addition to sample collection, certain anthropometric
measurements (height and weight) necessary
to calculate BMI were also measured (Table 1,).

Also Participants had to complete a self-administered
questionnaire by providing information regarding
their reproductive history and whether or not they
routinely practice any physical activity. If they did,
they were asked to fill the description, type, frequency,
intensity and duration of the physical activities
practiced. This information was used to calculate the
physical activities MET using the “Compendium of
physical activities” as proposed by Ainswort et al. (39)
which provided a measurement of their intensity level.

Conventional semen analysis

After complete liquefaction of the semen samples
(30-60 minutes, at 37°C), a basic semen
analysis was performed according to the World
Health Organization (WHO) guidelines (1) while
the sperm concentration was determined by using
a Makler chamber (Seﬁ-Medical Instruments,
Haifa, Israel) (54). Finally, sperm morphology was
analyzed following the Tygerberg strict criteria
(55), and semen samples with leukocytospermia
(>1×106 white blood cells/mL) were excluded.

Functional analysis

All flow cytometry analysis reported in this study
were conducted on an Epics XL flow cytometer
(Becton Dickinson, CA, USA) with a 488 nm excitation
wavelength supplied by an argon laser. Forward
scatter and side scatter measurements were
used to gate spermatozoa’ and exclude debris’ and
aggregates limiting undesired effects in the overall
fluorescence. All data were acquired and analyzed
using WinMDI 2.9 Software (Scripps Research Institute,
La Jolla, CA) and a total of 10000 events
were collected per sample.

Intracellular reactive oxygen species production

The intracellular ROS and RNS (specifically
H2O2, HO-, ROO- and ONOO-) levels were evaluated
using 2’, 7’-dichlorodihydrofluorescein diacetate
(DCFH-DA, Sigma-Aldrich, St Louis, MO,
USA). Upon cleavage of the acetate groups by intracellular
esterases, DCFH is selectively oxidized
by the above mentioned ROS and RNS to the green
fluorescent DCF. PI was used to exclude the necrotic/
dead cells (5). DCFH-DA was diluted to a final
concentration of 1 μM in 300 μL of PBS containing
2×106 spermatozoa and PI (final concentration
12 μM). The cell suspensions were incubated in
the dark for 5 minutes, at 25°C, washed three times
with PBS (180 x g, 5 minutes) and the pellet re-suspended
in PBS before being analyzed by flow cytometry.
Results are expressed as the percentage of
live spermatozoa exhibiting the green DCF fluorescent
response (DCF positive spermatozoa), as well
as the green media fluorescence intensity (MFI).

Plasma membrane integrity evaluation

The LIVE/DEAD® Sperm Viability Kit (Molecular
Probes Inc., The Netherlands) which distinguishes
three populations of sperm based on their
staining patterns, was used to assess the integrity of
the plasma membrane according to the manufacturer’s
instructions. Briefly, 2×106 spermatozoa were
incubated in 300 μL of PBS with Sybr-14 and PI
(green and red fluorescence emission, final concentration
of 1 μM and 12 μM, respectively) in the dark
(30 minutes, 25°C), washed once and re-suspended
in PBS prior to flow cytometry analysis. Data are
expressed as the percentage of viable spermatozoaintact
plasma membrane cells (positive to SYBR-14
and negative to PI), necrotic/dead cells (positive for
PI only) or moribund sperm (positive for both dyes).

Lipid peroxidation assay

Oxidative degradation of lipids was measured using
the BODIPY (581/591) C11 (Molecular Probes
Inc., The Netherlands) according to the method proposed
by Aitken et al. (16). BODIPY (581/591) C11
once incorporated into sperm membranes, undergoes
a fluorescent emission shift from orange to green
upon peroxidation by ROS. Briefly, 2×106 spermatozoa
suspended in 300 μL of PBS were incubated in
the dark (30 minutes, at 25°C) with BODIPY C11 (final
concentration 6.6 μM), washed and re-suspended
in PBS before flow cytometry analysis. Results are
expressed as the percentage of spermatozoa exhibiting
the green fluorescence response.

Statistical analysis

The distribution of the data was evaluated with
the normality test of residuals. The t test was used
to compare groups of data that assumed Gaussian
distribution, while the Mann-Whitney test
used to compare the variables that did not assume
Gaussian distribution. Correlations between sperm
variables were determined with the Pearson correlation
coefficient. Data were analyzed by using
Prism 5.0 (GraphPad Software, San Diego, CA)
statistical software and a P<0.05 considered to be
significant. Data following Gaussian distribution
are expressed as the mean ± SD and those not assuming
Gaussian distribution are expressed as median
and range.

Results

According to the MET scores, men were stratified
into a physically active group (PAG, 8-48
MET, n=17) and a sedentary group (SG, <3 MET,
n=15). Both PAG and SG present similar characteristics
with regards to abstinence (4.1 ± 0.69 vs.
3.7 ± 0.75 days), height (1.74 ± 0.06 vs. 1.72 ±
0.05 m) and BMI (23.7 ± 1.5 vs. 22.7 ± 1.8 kg/m2).
The average weight was slightly higher in the PAG
in comparison with SG (71.6 ± 7.3 vs. 67 ± 5.3
kg), because these men had increased body mass
in the form of muscle not of fat (Table 1,).

All semen samples from the PAG appeared
normal with regards to viscosity and showed no
agglutination, however various samples from
SG showed moderate to high viscosity (33%) as
well as isolated agglutination (47%) and moderate
to abundant agglutination (13%) respectively.
Among the conventional sperm parameters, total
sperm motility, progressive motility and the percentage
of viable sperm were significantly higher
(P<0.05) in the PAG compared to the SG (Table
2). The only functional parameter that showed significant
difference (P<0.05) between the PAG and
SG was the percentage of moribund spermatozoa
(Table 3,).

When comparing the combined data sets from
both groups, significant correlations were found
between total abnormal sperm forms and spermatozoa
with head defects (correlation coefficientr-= 0.67, P<0.01), sperm with neck/middle piece
defects and progressive motility (r=-0.56, P<0.01),
ejaculation abstinence time and sperm with excess
residual cytoplasm (r=0.58, P<0.01), ejaculation
abstinence time and non-progressive motility (r=-0.57, P<0.01), viable sperm and intracellular ROS
production (r=0.79, P<0.01), viable sperm and
sperm with high mitochondrial membrane potential
(ΔΨm, r=0.83, P<0.01), and sperm with high
ΔΨm and intracellular ROS production (r=0.65,
P<0.01). In addition, when the PAG’s data were
analyzed separately, all of the above mentioned
significant correlations were found, together with a
few significant correlations exclusive to the PAG.
These include: ejaculation abstinence time and immotile
sperm (r=-0.57, P<0.01), sperm concentration
and normal morphology (r=0.72, P<0.01), and
sperm concentration and sperm with head defects
(r=-0.63, P<0.01).

Discussion

found differences in conventional and functional
seminal parameters between physically active
group and sedentary group of men. The semen
parameters were better in PAG, which is in
favor to adopt such a life style. The average values
of the conventional parameters analyzed for each
group, remained above the lower limit reference
values proposed by the WHO (1). The total and
progressive sperm motility, sperm viability, as
well as the percentage of moribund cells were significantly
higher in the PAG compared to SG. This
is the first study in addition to conventional semen
parameters, certain sperm functional parameters
i.e. ΔΨm, plasma membrane integrity, intracellular
ROS and lipid peroxidation, were analyzed in relation
to the practice of vigorous physical activity
or following a sedentary lifestyle. Our results are
in accordance with previous study demonstrating
increased sperm motility in physical active men
(41) and comparable results in a group of assisted
reproduction patients classified according to their
physical status (43). However, significant differences
in sperm viability due to physical activity
levels had not been previously reported.

Some studies reported that sperm concentration
and morphology are the main parameters improved
in men having moderate to vigorous physical active
lifestyle (47), against being sedentary (43).
Our results are not in agreement with these findings,
as we did not observe any significant differences
in either sperm concentration or morphology
between PAG and SG. Nonetheless, a positive correlation
was found between sperm concentration
and normal morphology in PAG. Similar results
have been reported by Munuce et al. (57) in semen
samples obtained from men attending a reproductive
clinic without regarding their physical status.
This finding is interesting because it may be related
to an increase in hormones, specially folliclestimulating
hormone (FSH), luteinizing hormone
(LH) and testosterone, responsible to stimulate
proper spermatogonia nutrition and division during
the process of spermatogenesis (58, 59). This
speculation is supported by the findings from previous
studies where increased total and free blood
plasma and serum testosterone, as well as higher
FSH and LH levels have been demonstrated after
continuous moderate physical activity (41).

The plasma membrane integrity is a key determinant
for proper sperm interactions with other cells
and their environment, therefore it is a prerequisite
for successful fertilization (60). The percentage of
dual stained (moribund) spermatozoa was statistically
higher in SG in comparison with PAG. This
sperm population have been described as slightly
damaged sperm with compromised plasma membrane
that have lost their ability to exclude PI,
indicating a transitional phase in which the cell
ultimately die (60-62). Although the biological importance
of moribund sperm has not been well established,
in works on bulls the percentage of moribund
spermatozoa was positively correlated with
the low fertility status of males, possibly compromising
the availability of live sperm in the female
reproductive tract (62). Furthermore, Garner and
Johnson (61) have microscopically observed that
the change from green to red fluorescence of some
sperm, began at the posterior portion of the sperm
head, proceed anteriorly and is accompanied by
the progressive loss of motility until they are dead.
This increased percentage of moribund spermatozoa
and negative correlation between sperm with
neck/middle piece defects and progressive motility in SG can be the possible explanations of significantly
lower progressive and total motility in
these men.

The evaluation of functional parameters has also
been used to determine the levels of oxidative
stress in spermatozoa. Common sedentary activities
such as sitting for long, and some physical activities
including running or bicycling may disrupt
the intrascrotal temperature regulation (63, 64)
and increase the pressure force to the testicles (46,
47, 49), leading to oxidative stress (58).

Although the percentage of DCF positive spermatozoa
in the PAG group was higher in SG, it
was not significantly different. We found higher
values of DCF positive sperm in comparison with
previous studies (65-67) intended to evaluate the
ROS/RNS production on spermatozoa using the
same method. However; the oxidative stress level
as depicted by lipid peroxidation measurement was
discernibly lower in PAG than SG, which is in accordance
with previous findings by others (16, 68).
Sperm DNA integrity did not differ significantly in
our study between PAG, SG and DFI remained in
the range considered normal (16-24%) (69). This
is in accordance with a previous report where no
relation of sperm DFI was drawn in men with sedentary
lifestyle in relation to their BMI and their
waist circumference (70).

In addition, no mitochondrial dysfunction was
detected either in the PAG or SG group despite the
total and progressive motility is significantly increased
in PAG. In fact, most of the spermatozoa
in the semen samples from both groups had high
ΔΨm, which is indicative in proper mitochondrial
functioning (17, 71). It may support the assumption
that the rapid transition from viable to moribund
sperm was influencing the loss of motility
in the SG sperm rather than the viable sperm that
have diminished the ΔΨm as it may be commonly
related. As the higher ROS detection in the PAG
was not correlated with oxidative stress generation
in spermatozoa (higher lipoperoxidation-LPOand/
or altered DFI), we speculate that there must
have been a balance between pro-oxidants and
antioxidants molecules in the PAG volunteers’
semen samples. Possibly the practice of vigorous
physical activity of volunteers, have contributed
to attenuate the oxidative stress events in consequence
of the higher ROS/RNS production, since
it has been previously demonstrated that physical
training promotes blood total antioxidant capacity
(72), and also in semen, moderate to vigorous
physical activity practitioners had superior levels
of antioxidant enzymes in comparison with high
performance-elite athletes or sedentary men (73).

Furthermore; it is known that sperm cells have a
deficient ROS-scavenging system, in consequence
of its limited cytosolic space. So they are very dependent
on the antioxidant protection provided by
the male reproductive tract (74). This is directly
influenced by the men’s nutritional status and the
dietary intake of antioxidant molecules since they
form an essential part of the human antioxidant defense
system (75).

As we did not control the diet in our volunteers,
the effect of the diet cannot be ruled out, considering
that, a physically active lifestyle is commonly
accompanied by a healthy diet. In the light of these
results, we consider convenient to include some
other informational aspects, certainly related to
the physically active or sedentary lifestyle and the
semen quality. For instance, nutritional aspects related
with dietary antioxidants intake, the determination
of blood hormonal levels (mainly LH, FSH
and testosterone) and the semen total antioxidant
capacity evaluation, directly involved in the developmental
environment of spermatozoa and the
oxidative stress dynamics. On the other hand, it
has also been established that if the physical training
is at least moderate but regular, it may turn into
an adaptation to diminish the increased amounts of
ROS producing during high oxygen consumption
derived from further vigorous physical activities
(41, 58, 76, 77). This physical activity linked-adaptation
constitutes an advantage over the possible
adverse conditions associated with the practice of
some previously mentioned physical activities that
may negatively affect the seminal quality.

Infertility affects approximately 15% of couples
of reproductive age, with significant impact on their
quality of life (11, 70). As it is estimated that men
contribute equally (50%) to the causes of fertility
problems (29, 59), the identification and modification
of some potential risk factors such as the relationship
between physical activity or inactivity and
semen quality, may help some couples to achieve
their reproductive goals (70). The practice of vigorous physical activity is clearly not the unique solution.
Most of the literature regarding the relatioship
between physical activity, sedentarism and semen
quality, have focused on elite athletes or men attending
fertility clinics. However; various investigations
have demonstrated the positive influence of moderate,
constant exercise on the hormonal profile (41,
58, 76), libido (78), the psychological wellbeing
(59) and on the body condition (30, 38, 76), which
may also impact positively the male reproductive
outcome. Our volunteers may be clasified as recreational
but vigorous physical activity practitioners,
since none of them were endurance sport competitors
and the activities performed included strenght
and aerobic training or vigorous occupational physical
activities. This is important to clarify because
the type of physical training, specially the higher
intensity or constantly anaerobic training have been
related to diminishseminal parameters (45-47, 49)
and also may influence the hormonal effect on the
sperm quality, specially on the testosterone metabolism
(41, 59).

Conclusion

Despite the fact that some indicators of cellular
oxidative stress were higher in the PAG in comparison
with the SG, no signs of developing a state
of oxidative stress was observed. On the contrary,
the practice of vigorous physical activity in the
conditions set in our study (8 to 48 MET, in sessions
of two hours minimum, with a frequency of
at least 3 days a week), was significantly related to
better semen parameters (increased viability, progressive
and total motility and lower percentage
of moribund cells), when compared to individuals
following a complete sedentary lifestyle at least
for a year. It can therefore be concluded that the
levels of physical activity reported in this study,
exert a positive effect on the semen parameters of
these men or at least prevent its deterioration as a
result of environmental stressors. Our findings are
encouraging since they contribute to elucidate the
proper intensity and frequency of physical activity
which may excerpt a positive effect on semen
quality or at least prevent its decline related to the
practice of higher intensity-endurance physical activities.
Future studies are required in defining the
intensity and threshold to be considered as beneficial
for semen quality.

Acknowledgements

This study was financially supported by the sustainability
strategy (Reproduction Group) and Investigation
Center of Exact and Natural Sciences
(CIEN) of the University of Antioquia. The authors
report no declaration of interest.